Chloroquine use in the Treatment of COVID-19: Systems Biology Report of Common Targets of SARS-CoV-2 and Chloroquine.

BACKGROUND: Chloroquine use for treatment of COVID-19 patients has been under discussion and recommendations have been shifting from positive to caution or non-conclusive. Variability of clinical outputs requires understanding of mechanisms of the differences. Implementation of a companion diagnostic would allow selecting patients who may benet from the drug. The rst line would be markers already used in clinics. Systems biology opens for an opportunity to identify targets common for chloroquine and SARS-CoV-2. These common targets would be candidates for the companion diagnostic. METHODS: Systemic analysis of molecular mechanisms and markers engaged by chloroquine and SARS-CoV-2 virus was performed. The networks of regulatory mechanisms were explored for an intersection and relevance to clinical markers. RESULTS: Reported here systemic analysis describes the intersection of molecular mechanisms of chloroquine and processes engaged by COVID-19. 266 nodes provide insight into the mechanisms of chloroquine impact on the infection and represent a pool of companion diagnostic markers. As an example, an intersection with the markers of heart arrhythmia retrieved 19 nodes. Thirteen of them were reported in human plasma: levels of albumin, amyloid precursor protein, and endoglin correlate with adverse cardiac effects. CONCLUSIONS: Reported intersection nodes of SARS-CoV-2 and chloroquine are the candidate markers for companion diagnostic of the chloroquine application. Some of these markers are already used in the clinic and their interpretation may contribute to monitoring for adverse effects of chloroquine.


Background
The use of chloroquine for the treatment of COVID-19 patients has been under discussion (1)(2)(3). To be effective, chloroquine has to act on its targets that would lead to a therapeutic response. To discriminate between responding, non-responding and adverse effects-prone patients, there is a need of a companion diagnostic for chloroquine. Such markers are routine in oncology (4,5). These markers inform clinicians whether a drug would be useful for a given patient. Without these markers, an effect of drugs is frequently non-conclusive when evaluated at the population level. That may explain recent reports of non-conclusive bene t from use of chloroquine and hydroxychloroquine (www.who.int/publications/m/item/targetedupdate-safety-and-e cacy-of-hydroxychloroquine-or-chloroquine-for-treatment-of-covid- 19). Chloroquine and hydroxyl chloroquine are used as immunomodulators and showed promising data in in vitro studies of COVID-19 management (1)(2)(3). However, the number of clinical evidence is still not su cient to claim a high certainty conclusion. The systems biology approach may offer the way to identi cation of markers that would identify patients who may bene t from the drug and those patients who would not. Companion diagnostics with the predicted markers allows selection of responsive patients and prediction of the disease development.
Chloroquine has been used since the 1940th. Studies of this remedy generated information about molecular mechanisms of its action. An international DrugBank depository (www.drugbank.ca) is an example of a curated and proven drug target database (6). The studies of COVID- 19 are not yet as extensive as studies of chloroquine, but there are already reports of SARS-CoV-2 targets in human cells (7)(8)(9). Identi ed targets re ect molecular mechanisms engaged by chloroquine and COVID-19, and systems biology allows identi cation of these regulatory processes. A number of network building tools and high-quality databases are available for systemic analysis of molecular mechanisms engaged by COVID-19 and chloroquine (7,(10)(11)(12)(13)). An analysis of regulatory networks is the most comprehensive way to explore mechanisms that are initiated or dependent on the targets of COVID-19 and chloroquine. Comprehensiveness is ensured by the incorporation of the experimental data from hundreds to thousands of reports. For example, UniProt database contains 562,755 records of experimental data (uniprot.org) (14). This a rich source for systemic network analysis.
COVID-19 infection manifests in many different clinical symptoms (15)(16)(17). It indicates that the virus employs different molecular mechanisms and attacks different types of cells. Here we report an identi cation of potential markers to evaluate the e cacy of chloroquine in the treatment of COVID-19 patients. Our systemic analysis identi ed 266 nodes, i.e. genes and proteins that represent common molecular mechanisms engaged by chloroquine and COVID-19. An example of cardiac arrhythmia showed 19 potential companion diagnostic markers for chloroquine use and prediction of cardiac adverse effects.

Methods
The datasets for building networks were collected as follows, and are listed in Supplementary Table 1. For chloroquine, the targets were retrieved from the Drug Bank depository (drugbank.ca) (6). For SARS-CoV-2 interacting proteins, 322 interactors were reported by Gordon et al., and ACE2 and TMPRSS2 were used (7,18). For arrhythmia, markers described by Bose et al. were used (19).
The networks building and analysis was performed in Cytoscape (10). The signi cance for the inclusion of nodes and edges was set to p < 0.05. For the building of the networks, we used the UniProt database (14). For extraction of intersections, the "Network Analysis" tool of Cytoscape was used. Statistical signi cance of network building (inclusion of nodes and con dence of edges) was set on p < 0.05. BiNGO tool was used for the analysis of affected biological processes. For statistical signi cance, the level was set at p < 0.05, and the hypergeometric statistical test was used, with Benjamini and Hochberg false discovery rate correction.
A cross-validation analysis of identi ed nodes with published reports about their clinical values and a role in physiology was performed. We searched PubMed with the Medical Subject Headings (MeSH) of a node and words "COVID-19", "chloroquine", and "heart". Retrieved publications were scrutinized for information about clinical values of the nodes as markers and for involvement of the nodes in molecular mechanisms and biological processes of relevance for a virus infection, predictive marker value, correlation with clinical outputs and adverse effects, and a role in crucial intracellular regulatory mechanisms, e.g. proliferation, death and differentiation of cells.

Results
Identi cation of common targets of SARS-CoV-2 and chloroquine For chloroquine, there have been reported 11 direct targets, i.e. GSTA2, TNF, TLR9, GST, HMGB1, GSTM1, CYP2C8, CYP3A4, CYP3A5, CYP2D6 and CYP1A1 (Supplementary Table 1). Chloroquine impact on these targets may lead to engagement of a regulatory network containing 1,336 nodes and 2,526 edges (Supplementary Fig. 1; Supplementary File 1, network "Chloroquine_UniProt"). The network was built with the retrieval of interaction data from the UniProt database. The same database was used to build networks of angiotensin-converting enzyme 2 (ACE2) and type 2 transmembrane serine protease (TMPRSS2) and SARS-CoV-2 interactors that are listed in Supplementary Table 1 Table 2, Supplementary File 1, network "Intersection_ChloroqUniProt_CovUniProt_.."). This large number of common nodes indicates a signi cant molecular cross-talk between chloroquine and COVID-19. One hundred nine of these nodes were also detected in the human plasma (Table 1). These intersections identify mechanisms of chloroquine interference with SARS-CoV-2 action and list potential plasma markers (Fig. 2). The intersection nodes may represent markers of companion diagnostic for chloroquine use. If these nodes are affected in a patient infected with the virus, then the chloroquine prescription may be of help, as chloroquine would markers act on/via these affected nodes.

Covid-19 And Cardiac Arrhythmia Markers
To evaluate whether the intersection nodes would lead to the identi cation of clinically relevant markers, we used an example of cardiac arrhythmia. Markers of arrhythmia were used to generate a network ( Supplementary Fig. 4). The arrhythmia markers are OPN, ANXA5, GDF15, MPO, LGALS3, TNNT2, TNNI3, ANFB, REN, IL6 and CRP (Supplementary Table 1) (19). The arrhythmia network was explored further for the intersection with common nodes of chloroquine and COVID-19 regulatory mechanisms ( Fig. 1B; Supplementary File 1 network "Intersection_Arhythmia_Cov19_..")). There were no edges retrieved between these nodes and amyloid precursor protein was retrieved with 3 different accession numbers. We identi ed 19 nodes linking arrhythmia markers to chloroquine and COVID-19 ( Table 2). Analysis of these 19 nodes showed an engagement of processes affecting the heart and regulation of cell death and proliferation.
Detection of proteins in serum or plasma suggest their suitability as makers for repeatable sampling by blood collection. We used a database of proteins detected in plasma (http://www.plasmaproteomedatabase.org) and retrieved 13 proteins (Table 2). Then, we searched for reports of clinical applications of these 13 proteins as markers of cardiac conditions. Levels of human serum albumin (ALB), amyloid proteins (APP) and soluble endoglin (ENG) correlate with cardiovascular diseases (Fig. 2). It has to be noted that these markers have also been associated with general conditions and not only cardiac, e.g. hypoalbuminemia associated with liver and kidney diseases, or had a limited use in clinics, e.g. APP or ENG. Albumin concentration below 10 g/L correlates with cardiovascular diseases (20). Levels of amyloid precursor protein (APP) higher than 150 pg/mL correlate with cardiomyopathy (21). Amyloid-beta (1-40) protein was associated with the incidence of coronary heart failure (22). Two of other identi ed by us proteins, i.e. microtubule-associated protein tau (MART) and prion protein (PRNP) are also associated with the onset of cellular degeneration (23)(24)(25). Endoglin is involved in the development and regulation of vasculature. Elevated levels of soluble endoglin in plasma correlate with enhanced left ventricular lling pressure (26). 14-3-3zeta/delta (YWHAZ) is one of the 10 genes enhanced in ischemic stroke (27).
The systems biology approach allowed us to explore published original experimental data in the search for companion diagnostic markers for chloroquine. Reported here 109 intersection nodes represent a pool of these markers. The example of the search for markers to guide the use of chloroquine and preventing cardiac arrhythmia identi ed 19 candidates. Four of these were reported to correlate with adverse effects, thus con rming the potential clinical value of our approach. Monitoring of the described here markers may help in preventing severe side effects in COVID-19 patients, even if some of the markers are considered as general, or not-frequently used or even novel. The general (ALB) or not-frequently used (APP, ENG, MART, PRNP and YWHAZ) may be applied in clinics already now, as they are approved as markers. Novel candidate markers from the list of 19 nodes would have to be evaluated in clinical trials, and this work contributes with rationale for such trials.

Discussion
Systemic network analysis becomes a potent and e cient tool for the investigation of correlations and molecular mechanisms (8,12,13). Well-developed and curated databases contain large volumes of original experimental data. This data are available for analysis with a number of tools. Here, we used Cytoscape that allows retrieval of molecular interactions, functional dependencies, correlation and clinical data (10). Used by us the UniProt database contains more than 500,000 curated entries (14). This rich source of data in combination with the e cient analysis tool, i.e. Cytoscape, leads to unveiling novel dependencies. Two  Table 2). That may explain the clinical e cacy of chloroquine. However, changes in expression and/or activity of many of these nodes may also have undesirable consequences, leading to adverse effects of chloroquine. The complexity of chloroquine molecular mechanisms and differences in representation of these mechanisms in different individuals may lead to different clinical outputs.
This manuscript reports the identi cation of nodes (genes and proteins) common for SARS-CoV-2 and chloroquine. These interaction nodes may be in uenced by both the virus and the drug. Therefore, they would re ect whether and how chloroquine may in uence SARS-CoV-2-engaged mechanisms. Such nodes can be potential companion diagnostic markers of chloroquine, even if these markers are known for use for other clinical conditions. As an example of applicability of our data, we report 19 marker candidates for guiding chloroquine treatment of SARS-CoV-2-infected patients and monitoring for cardiac arrhythmia ( Table 2). Four of these markers are already known to affect cardiac conditions. The decrease in albumin to concentrations below 10 g/L correlates with cardiac adverse effects (20). Albumin levels have been recommended for clinical monitoring of COVID-19 patients (20,(28)(29)(30)(31). Hypoalbuminemia with the albumin levels lower than 35 g/L was associated with the 2-time higher risk of the long-term mortality in heart failure (31). Chloroquine was described as a drug against prion and Alzheimer's diseases (32). Prion protein and amyloid beta peptide are likely to be components of the innate immune system (33). Amyloid-beta protein association with coronary heart disease and amyloidosis-related heart disease was reported (21,22). Identi cation of amyloid precursor protein, microtubule-associated tau and prion proteins indicate a link of cell damage and degeneration to cardiac conditions.
Dab2 is involved in suppression of apoptosis by Epstein-Barr virus (EBV) (40). Two nodes, mitochondrial antiviral signaling protein (MAVS) and DExD/H-Box Helicase 58 (DDX58) were reported as antiviral proteins (41,42). Inhibition of MAVS expression decreased e cacy of hydroxychloroquine against dengue virus (42). These examples show that the identi ed nodes have a high probability to be markers for a companion diagnostic. The 19 markers annotated in Table 2 are the example of using the pool of 266 common nodes of COVID-19 and chloroquine. Our report provides a basis for further clinical studies of the potential markers.
Reported by us results can be used in clinical practice already now, as some of identi ed by us nodes are used in clinical diagnostics, e.g. albumin, or testing is available, even if not-frequently, e.g. soluble endoglin and amyloid precursor protein. These markers are used for non-COVID-19 conditions, and repurposing of their use for COVID-19 patients treated with chloroquine can be applied now. For example, a higher risk of adverse cardiac effects would be indicated by downregulation of albumin and up-regulation of amyloid precursor protein, tau protein, prion protein and soluble endoglin (21,22,26,43).

Conclusion
Presented here network analysis describes nodes common for SARS-CoV-2 and chloroquine. The common nodes are intersections of molecular mechanisms of the virus and the drug. Having two inputs, these nodes are potential markers of a companion diagnostic of chloroquine for the treatment of COVID-19 patients.

Consent for Publication:
Since, it is case report. Consent for publication is not applicable here. Personal or identifying information of study participants is not disclosed in any form in this paper.

Availability of data and materials
All datasets used for this study are available from corresponding author on reasonable request.

Competing interests
The authors declare no competing interests.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or not-for-pro t sectors. Work ow of selection of potential companion diagnostic markers. Two hundred sixty-six common COVID-19 and chloroquine nodes were evaluated for representation of biological functions and relevance to adverse effects. Retrieved with BiNGO tool biological processes and the nodes of the relevance to the heart arrhythmia markers are annotated.